RESUMO
FANCM is a DNA repair protein that recognizes stalled replication forks, and recruits downstream repair factors. FANCM activity is also essential for the survival of cancer cells that utilize the Alternative Lengthening of Telomeres (ALT) mechanism. FANCM efficiently recognizes stalled replication forks in the genome or at telomeres through its strong affinity for branched DNA structures. In this study, we demonstrate that the N-terminal translocase domain drives this specific branched DNA recognition. The Hel2i subdomain within the translocase is crucial for effective substrate engagement and couples DNA binding to catalytic ATP-dependent branch migration. Removal of Hel2i or mutation of key DNA-binding residues within this domain diminished FANCM's affinity for junction DNA and abolished branch migration activity. Importantly, these mutant FANCM variants failed to rescue the cell cycle arrest, telomere-associated replication stress, or lethality of ALT-positive cancer cells depleted of endogenous FANCM. Our results reveal the Hel2i domain is key for FANCM to properly engage DNA substrates, and therefore plays an essential role in its tumour-suppressive functions by restraining the hyperactivation of the ALT pathway.
RESUMO
Plant diseases seriously affect the growth of crops and the quality and yield of agricultural products. The search for plant-derived pesticide candidates based on natural products is a hot topic of current research. Marine natural products almazoles C-D were efficiently prepared and selected as the lead compounds in this work. Two series of almazole derivatives were designed and synthesized, and their antiviral and fungicidal activities were systematically evaluated. The results of anti-tobacco mosaic virus (anti-TMV) activity showed that almazoles C-D and their derivatives had good anti-TMV activities. Compounds 6, 15, 16a, 16b, 16g, 16l, 16n, 20a, 20d, 20i, and 20n exhibited better anti-TMV activities than the commercial antiviral agent ribavirin. Anti-TMV mechanism studies showed that compound 16b could induce the polymerization of 20S CP (coat protein, CP), thereby affecting the assembly of TMV virus particles. Molecular docking results showed that compounds 15, 16b, and 20n could combine with amino acid residues through hydrogen bonds to achieve an excellent anti-TMV effect. In addition, most of the almazole derivatives were found to have broad-spectrum fungicidal activities against eight kinds of plant pathogens (Fusarium oxysporum f. sp. cucumeris, Cercospora arachidicola Hori, Physalospora piricola, Rhizoctonia cerealis, Alternaria solani, Pyricularia grisea, Phytophthora capsici, and Sclerotinia sclerotiorum). This study provides an important evidence for the research and development of almazole alkaloids containing indole and oxazole structural groups as novel agrochemicals.
Assuntos
Produtos Biológicos , Vírus do Mosaico do Tabaco , Antivirais/farmacologia , Antivirais/química , Relação Estrutura-Atividade , Produtos Biológicos/farmacologia , Produtos Biológicos/química , Simulação de Acoplamento Molecular , Ribavirina/farmacologia , Desenho de FármacosRESUMO
Cancer cells establish replicative immortality by activating a telomere-maintenance mechanism (TMM), be it telomerase or the alternative lengthening of telomeres (ALT) pathway. Targeting telomere maintenance represents an intriguing opportunity to treat the vast majority of all cancer types. Whilst telomerase inhibitors have historically been heralded as promising anticancer agents, the reality has been more challenging, and there are currently no therapeutic options for cancer types that use ALT despite their aggressive nature and poor prognosis. In this Review, we discuss the mechanistic differences between telomere maintenance by telomerase and ALT, the current methods used to detect each mechanism, the utility of these tests for clinical diagnosis, and recent developments in the therapeutic strategies being employed to target both telomerase and ALT. We present notable developments in repurposing established therapeutic agents and new avenues that are emerging to target cancer types according to which TMM they employ. These opportunities extend beyond inhibition of telomere maintenance, by finding and exploiting inherent weaknesses in the telomeres themselves to trigger rapid cellular effects that lead to cell death.
Assuntos
Neoplasias , Telomerase , Replicação do DNA , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Telomerase/genética , Telômero/genética , Telômero/metabolismo , Homeostase do TelômeroRESUMO
BACKGROUND: Epithelial ovarian cancer (EOC) is the most lethal gynaecological malignancy with over 80% of cases already disseminated at diagnosis and facing a dismal five-year survival rate of 35%. EOC cells often spread to the greater omentum where they take-up cholesterol. Excessive amounts of cholesterol can be cytocidal, suggesting that cholesterol efflux through transporters may be important to maintain homeostasis, and this may explain the observation that high expression of the ATP-binding cassette A1 (ABCA1) cholesterol transporter has been associated with poor outcome in EOC patients. METHODS: ABCA1 expression was silenced in EOC cells to investigate the effect of inhibiting cholesterol efflux on EOC biology through growth and migration assays, three-dimensional spheroid culture and cholesterol quantification. RESULTS: ABCA1 suppression significantly reduced the growth, motility and colony formation of EOC cell lines as well as the size of EOC spheroids, whilst stimulating expression of ABCA1 reversed these effects. In serous EOC cells, ABCA1 suppression induced accumulation of cholesterol. Lowering cholesterol levels using methyl-B-cyclodextrin rescued the effect of ABCA1 suppression, restoring EOC growth. Furthermore, we identified FDA-approved agents that induced cholesterol accumulation and elicited cytocidal effects in EOC cells. CONCLUSIONS: Our data demonstrate the importance of ABCA1 in maintaining cholesterol balance and malignant properties in EOC cells, highlighting its potential as a therapeutic target for this disease.
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Pickering emulsions are eco-friendly, stabilized by solid particles, and have an essential role in leading industries. Although Pickering emulations have found several applications, surprisingly few investigations have attempted to explore the effectiveness of various mechanical processes for its production. To fill these gaps, the present investigation comprehensively examined the application of various Pickering emulsion preparation processes such as rotor-stator homogenization emulsification (R-SH), ultrasonic emulsification, and their combined processes by using nano-silica particles. The influences of emulsification time and intensity on emulsion droplets' distribution were analyzed as indicative factors. The kerosene/water nano-silica Pickering emulsion was utilized for all assessments. The obtained results demonstrated that the main distribution peak of the emulsion prepared by R-SH occurred where the chord length was greater than 40 µm. Micro-scale nano-silica-aggregates generated large droplets, while the fine-emulsion fraction was significantly increased after ultrasonic treatment. The experimental results showed that the emulsion prepared only by ultrasound needed substantial power to form a Pickering emulsion since the oil phase was difficult to disperse in the water phase. Finally, it was concluded that preprocessing by R-SH could form a stable and uniform emulsion speedily, which is essential for ultrasound emulsion preparation.
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Dióxido de Silício , Água , EmulsõesRESUMO
Approximately 25% of human neuroblastoma is caused by amplification of the MYCN oncogene, which leads to overexpression of N-Myc oncoprotein. The survival rate for this patient subtype is <50%. Here, we show that N-Myc protein bound to the DEAD-box RNA helicase DDX21 gene promoter and upregulated DDX21 mRNA and protein expression. Genome-wide differential gene expression studies identified centrosomal protein CEP55 as one of the genes most dramatically downregulated after DDX21 knockdown in MYCN-amplified neuroblastoma cells. Knocking down DDX21 or CEP55 reduced neuroblastoma cell cytoskeleton stability and cell proliferation and all but abolished clonogenic capacity. Importantly, DDX21 knockdown initially induced tumor regression in neuroblastoma-bearing mice and suppressed tumor progression. In human neuroblastoma tissues, a high level of DDX21 expression correlated with a high level of N-Myc expression and with CEP55 expression, and independently predicted poor patient prognosis. Taken together, our data show that DDX21 induces CEP55 expression, MYCN-amplified neuroblastoma cell proliferation, and tumorigenesis, and that DDX21 and CEP55 are valid therapeutic targets for the treatment of MYCN-amplified neuroblastoma.
Assuntos
Proteínas de Ciclo Celular/genética , RNA Helicases DEAD-box/genética , Neuroblastoma/genética , Animais , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/patologia , Regiões Promotoras GenéticasRESUMO
The ability of the N-MYC transcription factor to drive cancer progression is well demonstrated in neuroblastoma, the most common extracranial pediatric solid tumor, where MYCN amplification heralds a poor prognosis, with only 11% of high-risk patients surviving past 5 years. However, decades of attempts of direct inhibition of N-MYC or its paralogues has led to the conclusion that this protein is "undruggable." Therefore, targeting pathways upregulated by N-MYC signaling presents an alternative therapeutic approach. Here, we show that MYCN-amplified neuroblastomas are characterized by elevated rates of protein synthesis and that high expression of ABCE1, a translation factor directly upregulated by N-MYC, is itself a strong predictor of poor clinical outcome. Despite the potent ability of N-MYC in heightening protein synthesis and malignant characteristics in cancer cells, suppression of ABCE1 alone selectively negated this effect, returning the rate of translation to baseline levels and significantly reducing the growth, motility, and invasiveness of MYCN-amplified neuroblastoma cells and patient-derived xenograft tumors in vivo. The growth of nonmalignant cells or MYCN-nonamplified neuroblastoma cells remained unaffected by reduced ABCE1, supporting a therapeutic window associated with targeting ABCE1. Neuroblastoma cells with c-MYC overexpression also required ABCE1 to maintain cell proliferation and translation. Taken together, ABCE1-mediated translation constitutes a critical process in the progression of N-MYC-driven and c-MYC-driven cancers that warrants investigations into methods of its therapeutic inhibition. SIGNIFICANCE: These findings demonstrate that N-MYC-driven cancers are reliant on elevated rates of protein synthesis driven by heightened expression of ABCE1, a vulnerability that can be exploited through suppression of ABCE1.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/patologia , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Progressão da Doença , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Proteína Proto-Oncogênica N-Myc/metabolismo , Biossíntese de Proteínas , RNA MensageiroRESUMO
Epithelial ovarian cancer (EOC) is a complex disease comprising discrete histological and molecular subtypes, for which survival rates remain unacceptably low. Tailored approaches for this deadly heterogeneous disease are urgently needed. Efflux pumps belonging to the ATP-binding cassette (ABC) family of transporters are known for roles in both drug resistance and cancer biology and are also highly targetable. Here we have investigated the association of ABCC4/MRP4 expression to clinical outcome and its biological function in endometrioid and serous tumors, common histological subtypes of EOC. We found high expression of ABCC4/MRP4, previously shown to be directly regulated by c-Myc/N-Myc, was associated with poor prognosis in endometrioid EOC (P = .001) as well as in a subset of serous EOC with a "high-MYCN" profile (C5/proliferative; P = .019). Transient siRNA-mediated suppression of MRP4 in EOC cells led to reduced growth, migration and invasion, with the effects being most pronounced in endometrioid and C5-like serous cells compared to non-C5 serous EOC cells. Sustained knockdown of MRP4 also sensitized endometrioid cells to MRP4 substrate drugs. Furthermore, suppression of MRP4 decreased the growth of patient-derived EOC cells in vivo. Together, our findings provide the first evidence that MRP4 plays an important role in the biology of Myc-associated ovarian tumors and highlight this transporter as a potential therapeutic target for EOC.
Assuntos
Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/patologia , Genes myc/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Carcinoma Endometrioide/genética , Carcinoma Endometrioide/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Prognóstico , RNA Interferente Pequeno/genética , Taxa de SobrevidaRESUMO
The majority of patients with neuroblastoma due to MYCN oncogene amplification and consequent N-Myc oncoprotein over-expression die of the disease. Here our analyses of RNA sequencing data identify the long noncoding RNA lncNB1 as one of the transcripts most over-expressed in MYCN-amplified, compared with MYCN-non-amplified, human neuroblastoma cells and also the most over-expressed in neuroblastoma compared with all other cancers. lncNB1 binds to the ribosomal protein RPL35 to enhance E2F1 protein synthesis, leading to DEPDC1B gene transcription. The GTPase-activating protein DEPDC1B induces ERK protein phosphorylation and N-Myc protein stabilization. Importantly, lncNB1 knockdown abolishes neuroblastoma cell clonogenic capacity in vitro and leads to neuroblastoma tumor regression in mice, while high levels of lncNB1 and RPL35 in human neuroblastoma tissues predict poor patient prognosis. This study therefore identifies lncNB1 and its binding protein RPL35 as key factors for promoting E2F1 protein synthesis, N-Myc protein stability and N-Myc-driven oncogenesis, and as therapeutic targets.
Assuntos
Carcinogênese/genética , RNA Longo não Codificante/metabolismo , Proteínas Ribossômicas/metabolismo , Animais , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Fator de Transcrição E2F1/metabolismo , Feminino , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/genética , Neuroblastoma/patologia , Prognóstico , Biossíntese de Proteínas , Estabilidade Proteica , RNA Longo não Codificante/genética , Transcrição Gênica , Regulação para Cima/genéticaRESUMO
Survival rates for pediatric patients suffering from mixed lineage leukemia (MLL)-rearranged leukemia remain below 50% and more targeted, less toxic therapies are urgently needed. A screening method optimized to discover cytotoxic compounds selective for MLL-rearranged leukemia identified CCI-006 as a novel inhibitor of MLL-rearranged and CALM-AF10 translocated leukemias that share common leukemogenic pathways. CCI-006 inhibited mitochondrial respiration and induced mitochondrial membrane depolarization and apoptosis in a subset (7/11, 64%) of MLL-rearranged leukemia cell lines within a few hours of treatment. The unresponsive MLL-rearranged leukemia cells did not undergo mitochondrial membrane depolarization or apoptosis despite a similar attenuation of mitochondrial respiration by the compound. In comparison to the sensitive cells, the unresponsive MLL-rearranged leukemia cells were characterized by a more glycolytic metabolic phenotype, exemplified by a more pronounced sensitivity to glycolysis inhibitors and elevated HIF1α expression. Silencing of HIF1α expression sensitized an intrinsically unresponsive MLL-rearranged leukemia cell to CCI-006, indicating that this pathway plays a role in determining sensitivity to the compound. In addition, unresponsive MLL-rearranged leukemia cells expressed increased levels of MEIS1, an important leukemogenic MLL target gene that plays a role in regulating metabolic phenotype through HIF1α. MEIS1 expression was also variable in a pediatric MLL-rearranged ALL patient dataset, highlighting the existence of a previously undescribed metabolic variability in MLL-rearranged leukemia that may contribute to the heterogeneity of the disease. This study thus identified a novel small molecule that rapidly kills MLL-rearranged leukemia cells by targeting a metabolic vulnerability in a subset of low HIF1α/low MEIS1-expressing MLL-rearranged leukemia cells.